In 1966, Ishizaka established that the human allergen reaginic antibodies belong to a distinct class of immunoglobulins, IgE. There followed a decade of remarkable sequence of events. Sensitization of the human and monkey skin to Prausnitz-Kustner reaction by IgE antibodies was demonstrated. It was recognized that the role of IgE is central to the release mechanisms of chemical mediators of anaphylaxis; it mediates immunologic release of histamine from human leucocytes and mast cells; it primes the human lung tissue for antigen-induced release of histamine and Slow Reacting Substance of Anaphylaxis; it triggers the release of eosinophil chemotactic factor from human lung; and its affinity for attachment, through its Fc portion, to the receptors on the surface of mast cells and basophil granules was shown. It has been inferred that the number and the affinity of IgE antibodies bound to the basophil granulocytes determines the sensitivity of this cell to the allergen, while the histamine release induced by the antigen-antibody reaction on the cell surface is the function of the intracellular enzyme system and cyclic AMP level.
Dating back to the early part of the 20th century, patients with hay fever were treated with injections of incriminated allergens, albeit without understanding the pathologic bases of the disease or the pharmacologic bases for the efficacy of the therapy. Johansson's observation, in 1967, of augmented levels of serum IgE in atopic patients sparked intense interest in this relationship. Individuals with inhalant allergies were found to display seasonal peaks in their serum IgE levels; abatement of allergic symptomatology with immunotherapy was documented. Partial suppression of seasonal peaks following specific immunotherapy was demonstrated and the interrelationship of levels of IgE and IgG in atopic subjects, and the changes induced by specific immunotherapy have been illuminated.
The major in vitro test used to determine IgE today is a radioimmunoassay technique known as the Radio Allergo Sorbent Test or RAST. A major improvement in this technique is described in Nalebuff U.S. Pat. No. 4,243,641.
The advent of enzyme-labeled antibodies has been a major event in the progress of immunoassays. The use of such enzyme labels, for this purpose introduced in 1971, offers several advantages over radioimmunoassay techniques including the freedom from hazards of radioactive material, the stability of a label for months and possibly longer, the use of photometric rather than radiometric equipment, and at times, elimination of separation procedures. For these reasons, enzyme immunoassays have found widespread and diversified application both in reasearch and in clinical practice.
In 1966, Nakane & Pierce published a report demonstrating that peroxidase could be coupled to an antibody by a simple procedure to produce a stable conjugate. The intact immunological reactivity of such a conjugate was shown to render it eminently suitable for use in immunotracing methods, in a fashion similar to that of fluorescein-labeled antibody. Since that time a number of conjugates have been developed and used for enzymatic immunological tests. See, e.g., U.S. Pat. Nos. 4,016,043 and 3,645,852.
For the assay of total IgE in serum, alkaline phosphatase has been employed as the enzyme marker in an application of the enzyme linked immunosorbent assay and in a magnetic enzyme immunoassay. The use of both alkaline phosphatase and galactosidase has been described for the assay of allergen-specific IgE antibodies. The success in terms of quantitation, sensitivity and absence of non-specific reaction in any solid phase immunoenzymatic technique depends, to a great extent, on the quality of the enzyme-antibody conjugate. The immunoenzymatic techniques described in the literature for use with IgE have been performed using conjugate prepared by means of bifunctional reagents. Thus, the use of alkaline phosphatase (molecular weight 60,000) or galactosidase (molecular weight 580,000) as enzyme markers has required the use of glutaraldyde for conjugation of the enzyme to the antibody. The negative controls of such procedures exhibit a high degree of background activity or interference which interferes with the readability, i.e., interpretation, of the test results. While no precise quantitation has been reported, we have found that the use of alkaline phosphatase and galactosidase as enzyme markers has resulted in the final yield of a very small amount of functionally usable conjugates, usually 30% or less, and unacceptable contamination with large amounts of side reaction products. Such by-products include enzyme-enzyme conjugates, IgG-IgG conjugates and large aggregates. The separation of the usable conjugate from the undesirable side reaction products has been tedious and time consuming and, in addition, the stability of the purified fraction has not been satisfactory.
Recently, a hetero-bifunctionaI reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) has been successfully used for the conjugation process. In this procedure, SPDP reacts specifically with primary amino groups of proteins to produce a 2-pyridyl disulfide derivative. One of the proteins is thiolated by an additional reduction step under conditions which do not affect native disulfide bonds. The two modified proteins are then linked by a disulfide bond. Using B-galactosidase conjugates of anti-human IgE prepared by the above procedure, enzyme immunoassays for the determination of total IgE and allergen-specific IgE have been developed.
An in vitro solid phase immunoenzymatic allergy test for the presence of IgE which is very successful in terms of quantification, sensitivity and absence of nonspecific reaction is described in our U.S. Pat. No. 4,256,833. In this test, the enzyme employed is horseradish peroxidase (HRPO, molecular weight 40,000) and the conjugate is prepared using the enzyme with its free amino groups blocked with phenylisothiocyanate as the first step and the reduction of the conjugate is carried out by titration.
An inherent weakness in the immunoassays for IgE are that these assays fail to prevent interference by antibodies which do not belong to the IgE class of immunoglobulins but have specificity for the particular allergen under study. Such antibodies are generally regarded as belonging to IgG and IgA classes of antibodies. For example, immuno therapy with pollen antigens and bee venom results in a gradual fall in the level of allergen specific IgE antibodies, a rise in allergen specific IgG and allergen specific IgA antibodies, and a decline in basophil responsiveness. Naturally occurring IgG and IgA antibodies with an affinity for various allergens also occur in patients with inhalant allergy. The presence in serum of these "blocking" antibodies has been demonstrated by two approaches. First, the serum containing these antibodies can inhibit the antigen-induced release of histamine from basophils of sensitive patients, and second, such serum can inhibit the RAST test after IgE in serum has been inactivated with heat. The competition of IgG and IgA antibodies with the allergen specific IgE antibodies for allergen linked to an immunosorbent has been shown to occur with microcrystalline cellulose as well as with filter paper discs used in the commercially available RAST test. In the second step of the RAST assay, the binding of a labeled antibody to allergen-specific IgE antibodies is proportionally reduced and this may result in underestimation of the quantity of specific IgE in the test sample.
The IgG and IgA antibody interference not only impairs the diagnostic efficiency of the RAST test and the enzyme immunoassays for specific IgE, but it also masks the impact of immunotherapy on the serum levels of specific IgE and IgG antibodies. It is well established that specific immunotherapy results in the production of allergen-specific IgG antibodies and the conventional RAST and enzyme immunoassay techniques are therefore likely to produce spuriously low results for serum levels of IgE antibodies in this setting. This may also occur in allergic patients with high titers of naturally occurring IgG and IgA antibodies with specificity for the allergen under investigation.
Similar problems are also encountered in the use of the enzyme-labeled antibodies in histochemical localization of antigenic substances in the tissues. The use of antibody-antigen reactivity for determining the presence of various substances in animal tissue is known. For example, if one were seeking to determine whether insulin was present in a given tissue sample, the tissue would be contacted with antibody for insulin. The two Fab portions of the antibody combine with the insulin in the tissue and the Fc part is available for binding to a second antibody against the immunoglobulin G of the first species. A labeled second antibody is then reacted with the test system and an analysis is made for the label. Unfortunately, a preparation of anti-insulin antibody in the easily available form is a purified IgG fraction which in addition to specific antibodies to insulin also contains, in excess, other unwanted IgG molecules which include heterophile antibodies, naturally occurring antibodies and the like. Such unwanted IgG molecules can have the ability to bind indiscriminately to tissue components and thus render the specific localization of insulin highly ambiguous.
Unambiguous localization of a given antigen by the principle outlined above is possible only through the use of antibodies purified by affinity chromatography, which at present is prohibitively expensive.
In our U.S. Pat. No. 4,256,833, we describe a stable peroxidase immunoglobulin conjugate which is used in an enzyme immunoassay in which an allergen is immobilized on a solid phase, the test serum is incubated with the immobilized allergen and the amount of specific IgE bound to the allergen is quantified by incubating it with the enzyme labeled immunoglobulin.
It is the object of this invention to provide a new and improved method and reagent for carrying out an immunoenzymatic test for the presence of allergen-specific IgE, for proteins not belonging to the immunoglobulin class and for drugs which have or can be made to have a primary amino group.
This and other objects of the invention will become apparent to those skilled in this art from the following detailed description.